‘The Right Effect’

Genetically modified (GM) yeasts have found their place in the ethanol industry with more than half of producers routinely using them. All yeast providers, plus enzyme providers expanding into yeasts, now offer GM yeasts in their portfolios. DuPont Industrial Biosciences has Synerxia, Lallemand Biofuels & Distilled Spirits has TransFerm, Leaf-LeSaffre has ER-Xpress, DSM has eBoost, Novozymes has Innova, Taurus Energy has XyloFerm.

“The fuel ethanol market was a logical place to introduce these types of yeast,” says Emily Stonehouse, research and development manager at Mascoma LLC, a division of Lallemand Inc. “This industry has allowed us to demonstrate the safety and build trust in the use of a GM yeast and I believe this will have an effect on the acceptance of GM yeast outside of fuel ethanol.”

“Yeast research has been going on for a very long time,” says Pauline Teunissen, principal scientist at Dupont Industrial Biosciences. “We do know a lot about yeast, but to be honest, every time you make a change, if you do genetic engineering, you will need to do some research to figure out what the effects are because it can have multiple reactions in a yeast cell.”

The research on yeast metabolism took off when it was sequenced in 1996—the first organism with a nucleus and DNA organized in chromosomes to be fully sequenced. Earlier work had been done with viruses and bacteria with simpler genetic structures. It took 92 laboratories around the world working for three years to sequence Saccharomyces cerevisiae. The work opened the way to learning how genes impact cell function and how metabolic pathways work, and led to yeast becoming the focus of multiple genetic engineering studies.

Sixteen years later, in 2012, the first GM yeasts entered the ethanol market when LBDS introduced TransFerm, a glucoamylase-expressing strain engineered with a single fungal gene, and added a glycerol-reducing pathway the following year. DuPont introduced Synerxia in 2013, and other companies quickly followed. Today, the speed and cost of genome sequencing has improved to the point where any strain can be sequenced in less than a week at a cost around $3,000. Plus, new genetic engineering tools are promising to speed up the process of modifying and testing new strains.

“Genetic engineering has changed rapidly,” Stonehouse says. “Techniques used to be much less precise.” Work to add enzyme expression, for example, involved adding DNA extracted from an enzyme-producing organism into the chromosome of yeast and then screening the strains for high expression of the enzyme. The early techniques often used antibiotic markers that later had to be removed.

Introducing foreign genes is one technique, Teunissen says. Other techniques could be considered an acceleration of evolution. “The techniques involve putting yeast in a certain environment where you can show certain genes are expressed more than others, and then you actively work on those genes to turn them on or off, as needed.”

To do the work, researchers rely on yeast genome libraries and their own collections of yeast strains that can number in the thousands. “Because the genome has been sequenced for yeast, and we know each gene that is present, we can compare our own new yeast strain with the publicly known yeast genome,” Teunissen says. “We may see 50 differences, and then we zoom in to find out which are most important. And in some cases, it’s not just one gene, but multiple genes that work together to get the right effect.”

How it WorksYeast genetics involve about 6,000 genes comprised of combinations of adenine, thymine, cytosine and guanine (ATCG), sugar-phosphate molecules with a different amine group hanging off each one that become the basic building blocks of DNA. In sequencing, colored dyes enable a computer to read the ATCG sequence. In genetic engineering, enzymes are used to copy or chemically synthesize the DNA strands from a related yeast, fungi or bacteria with a desired trait. Those DNA fragments are introduced to the yeast chromosomes in the cell nucleus. Then the work begins to determine whether the desired trait was successfully transferred and how the other parts of the yeast metabolism responded.

Just as the cost and speed of sequencing have been dramatically reduced, the time and cost for genetic engineering is dropping. One of the new tools getting a lot of attention is CRISPR-CAS9. (CRISPR stands for clustered regularly interspaced short palindromic repeats). In researching the function of those unique CRISPR sequences in bacteria, scientists determined the CAS9 enzyme action was important in the microorganism’s immune system. When attacked, the bacteria produce enzymes to destroy a virus by cutting up its genetic material, storing some of the virus’ genetic code to be used to identify and enzymatically destroy similar viruses. By feeding manufactured code to the CRISPR sections, the researchers learned they could target a specific spot on a chromosome, knocking out the targeted gene and enabling a gene substitution, if desired.

“CRISPER can do in one step what took multiple steps and multiple days with the old technique,” Teunissen says. The cost of the procedure has also dropped from around $20,000 to less than $50.Examining all the effects of genetic changes is important, Teunissen says. “We cannot always predict which genes are turned on or off, so we need to test that.” In adding a metabolic pathway to yeast using three enzymes to more efficiently convert glucose to ethanol, she says, the yeast began making different cellular byproducts. When changes like that are found, the researchers have to evaluate whether the impact is positive, negative or neutral. In the case of glycerol reduction strategies, it’s important to remember that glycerol contributes to yeast health, she says, and diverting too much into ethanol production might result in yeast cells that can’t handle the stresses later in the fermentation cycle. “Whenever you change something, you will need to test for the other effects,” Teunissen says. “What I notice when I talk to ethanol producers, particularly in the beginning when they began using GM yeast, was that they are hoping for something that fits into the process, so they don’t have to make changes,” Teunissen says. There needs to be a willingness to tweak process conditions to optimize for the new yeast, which might involve adjustments to pH, temperature staging or enzyme dosing.

The success of a new yeast is dependent upon the interaction of the biotech team and technical sales and service team, Stonehouse says. “We have to understand what the real process is and how to scale our laboratory testing of new yeasts to more process-relevant testing.” The technical service team also works with plants to tailor the process to realize the value of the new yeast, she says. “It can be small tweaks, often it’s the timing of things.”

Care and Good Science “The competition among the companies is driving innovation,” Stonehouse says, and she notes that groups are already moving beyond glycerol reduction and glucoamylase expression to look at other enzymes and other metabolic enhancements.

Engineering new yeasts goes beyond evaluating their performance in fermentation to ensuring the acceptability of the feed coproduct. In introducing TransFerm, Stonehouse says Lallemand went the full regulatory route of convening a panel of independent experts to evaluate their scientific work. Animal feeding trials involved not only feed performance, but whether there were any impacts on the meat or milk. Getting GRAS status (generally recognized as safe) and a feed definition with the American Association of Feed Control Officials were important, though time-consuming, steps.

The advances in genetic engineering are having an impact here as well, Stonehouse says. “The regulatory agency can ask us for the full genome sequence. It’s a powerful tool to make sure the targeted change was done. Sequencing is a powerful tool to layer on top of genetic engineering to gain knowledge, but also to build trust in what you’ve done.” It’s important, she says, “that the industry understands the care that goes into the development of the new yeasts and the good science behind them.”